Method for measuring ethanolamine phosphate

ABSTRACT

A measurement method of ethanolamine phosphate includes a first step of adding an enzyme, which can catalyze a reaction that forms acetaldehyde from ethanolamine phosphate, to a sample, and conducting a first enzymatic reaction to form acetaldehyde, phosphoric acid and ammonia; and a second step of quantifying at least one of the resultant acetaldehyde, phosphoric acid and ammonia to determine an amount of the ethanolamine phosphate in the measurement sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Division of application Ser. No. 14/237,488 issuesas U.S. Pat. No. 9,631,224, filed Feb. 6, 2014, which is a NationalStage Application of PCT/JP2012/078749, filed Nov. 6, 2012, which claimsforeign priority from Japanese Patent Application Number 2011-246881,filed Nov. 10, 2011.

TECHNICAL FIELD

This relates to a measurement method of the ethanolamine phosphate levelin a sample.

BACKGROUND ART

Depression is a kind of mood disorder, and is accompanied by “depressedmood” and “loss of interest or pleasure” as primary symptoms. Accordingto a survey among medical institutions in Japan, more than 700,000depressive patients were considered to exist in 2008. However, adiagnosis of depression depends in a large part on the subjective viewof a physician or psychologist about emotional aspects of a patient oron the subjective view and response of the patient himself or herself,and is hardly considered to be made through an objective determination.Attempts have, therefore, been made in recent years to identify acomponent in a body fluid of a patient as an objective guideline for thediagnosis of depression.

It has been reported to be able to determine, as a predictive marker fordepression, the level of triptophan or its degradation product, theexpression level of a specific gene, or the like in a body fluid (PatentDocuments 1 and 2). In the meantime, the present inventors found thatthe ethanolamine phosphate level in blood is useful as a biomarker fordiagnosing depression (Patent Document 3).

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: WO-A-2006/105907-   Patent Document 2: JP-A-2008-253258-   Patent Document 3: WO-A-2011/019072

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

An ethanolamine phosphate level is highly reliable and excellent as abiomarker for depression, but its measurement has been limited to ameasurement method that uses a CE-TOFMS (capillary electrophoresistime-of-flight mass spectrometry) system. However, measurements byCE-TOFMS are time consuming (30 to 40 minutes). Moreover, CE-TOFMSsystems are expensive and precision analysis equipment. Because of this,CE-TOFMS systems are equipped in only a limited number of universitiesand analysis institutes. There is, accordingly, an outstanding need forestablishing a quick and simple measurement method, which allows evenlocal hospitals and clinics to measure an ethanolamine phosphate levelas a biomarker for depression.

Objects of the present invention are, therefore, to provide a method forsimply measuring the ethanolamine phosphate level in a sample, and also,a reagent, kit, program and the like useful in the method.

Means for Solving the Problem

The above-described objects can be achieved by the present invention tobe described hereinafter. Described specifically, the present inventionprovides a measurement method of ethanolamine phosphate, comprising afirst step of adding an enzyme, which can catalyze a reaction that formsacetaldehyde from ethanolamine phosphate, to a sample, and conducting afirst enzymatic reaction to form acetaldehyde, phosphoric acid andammonia; and a second step of quantifying at least one of the resultantacetaldehyde, phosphoric acid and ammonia to determine an amount of theethanolamine phosphate in the measurement sample.

The enzyme may preferably be an enzyme having a GabT domain.

Advantageous Effects of the Invention

According to the present invention, there are provided a method forsimply measuring the ethanolamine phosphate level in a sample and areagent useful in the method, and a diagnostic agent capable of simplydiagnosing depression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing ethanolamine phosphate (EAP)-degradingactivity of a Pan lysate.

FIG. 2 illustrates a graph showing analysis results of another Panlysate by anion chromatography (lower) and a graph showing SDS-PAGEresults of respective factions (upper).

FIG. 3 illustrates a graph showing analysis results of some fractions ofthe another Pan lysate by hydrophobic chromatography (lower) and a graphshowing SDS-PAGE results of respective sub-factions (upper).

FIG. 4 illustrates a graph showing analysis results of some otherfractions of the another Pan lysate through a hydroxyapatite column(lower) and a graph showing SDS-PAGE results of respective sub-factions(upper).

FIG. 5 is a graph showing measurement results of ethanolaminephosphate-degrading enzyme activity in GabT expression systems.

FIG. 6 is a diagram showing alignment results of GabTs of SEQ ID NOS: 1,2, 4, 5, 6, and 7.

MODES FOR CARRYING OUT THE INVENTION

In the method of the present invention for the measurement of anethanolamine phosphate level, an enzyme which may hereinafter be called“the first enzyme” is used in the first step. The first enzyme catalyzesa reaction, which hydrolyzes ethanolamine phosphate (CAS RegistryNumber: 1071-23-4) to form acetaldehyde, phosphoric acid and ammonia andwhich is represented by the below-described reaction equation (1). Thisreaction may hereinafter be called “the first enzymatic reaction”.

Heretofore, no enzyme has been identified yet to catalyze the one-stepreaction of equation (1). However, the present inventors isolated anenzyme having catalyst activity for the above-described reaction from aPantoea ananatis strain, and identified that enzyme to beγ-aminobutyrate aminotransferase (GabT).

In the measurement method of the present invention, at least one ofacetaldehyde, phosphoric acid and ammonia formed through the enzymaticreaction is quantified in the second step to determine the amount ofethanolamine phosphate in the sample. Only one of acetaldehyde,phosphoric acid and ammonia may be subjected to the quantification, butthe quantification of two or more of them makes it possible to reduce ameasurement error.

When quantifying acetaldehyde in the second step, it can be quantifiedwith good sensitivity by making use of the property of reducednicotinamide adenine dinucleotide (NADH) that it absorbs 340 nmwavelength ultraviolet rays well. Specifically, a method comprising thefollowing two sub-steps can be mentioned.

As the first sub-step, acetaldehyde dehydrogenase (ALDH) and oxidizednicotinamide dehydrogenase (NAD⁺) are added to the measurement sampleafter the above-described first step to oxidize acetaldehyde in thesample to acetic acid [see equation (2)].

In this reaction, NAD⁺ is reduced to NADH. Because only NADH stronglyabsorbs 340 nm ultraviolet rays, the measurement of the absorbance at340 nm of the measurement sample after the reaction as the next sub-stepcan lead to quantifying the amount of acetaldehyde formed in themeasurement sample after the first step, and from the value soquantified, the amount of ethanolamine phosphate in the measurementsample can be determined. Upon measurement of the acetaldehyde level inthe second step, a commercial acetaldehyde quantification kit (forexample, “F-Kit Acetaldehyde”, product of Roche Diagnostics GmbH) can beused.

In the first step, it is preferred to use a coenzyme in combination sothat the efficiency of the enzymatic reaction can be increased further.As a preferred coenzyme, pyridoxal phosphate can be mentioned.

No particular limitation is imposed on the enzyme for use in the firststep insofar as it can catalyze the reaction of equation (1) thathydrolyzes ethanolamine phosphate to form acetaldehyde, phosphoric acidand ammonia in one step. As examples of the enzyme having this enzymeactivity, enzymes having a GabT domain can be mentioned. The term “GabTdomain” means an amino acid sequence region defined byGXXXBADEBQXGFZRXG, wherein X means an amino acid, B denotes a branchedamino acid selected from the group consisting of I, L and V, and Zsignifies G or A.

As will be made apparent in examples to be described subsequentlyherein, the present inventors found that in a wide variety of organismspecies including archaebacteria, prokaryotes and eukaryotes such asplants and animals, their amino acid sequences have high homology of atleast 90% with the GabT domain of γ-aminobutyrate aminotransferase. Asexamples of the enzyme which has enzyme activity capable of catalyzingthe reaction of equation (1) and can be suitably used in the first step,proteins with amino acid sequences having at least 90% of homology withthe GabT domain can be mentioned.

Specific examples of the enzyme, which has enzyme activity capable ofcatalyzing the reaction of equation (1) and has a GabT domain, include aprotein (SEQ ID NO: 1 in the accompanying sequence listing) having aGabT domain derived from Pantoea ananatis and a protein (SEQ ID NO: 2)having a GabT domain derived from E. coli, both of which were identifiedby the present inventors. For use in the first step of the measurementmethod according to the present invention, the enzyme can be any proteininsofar as it has activity to catalyze the reaction of equation (1).Therefore, the enzyme may also be a protein consisting of an amino acidsequence similar to the amino acid sequence represented by SEQ ID NO: 1or 2 except that one or a few amino acids have been deleted, substitutedor added, or a protein consisting of an amino acid sequence having atleast 90% of homology with the amino acid sequence represented by SEQ IDNO: 1 or 2. The enzyme may be in an unpurified form, and even a partialpurification product or a cell lysate containing the enzyme is usableinsofar as it has the enzyme activity. As an example of the cell lysate,a supernatant obtained by the centrifugation of a Pantoea ananatislysate can be mentioned.

The method of the present invention for the measurement of anethanolamine phosphate level is very simple in procedure, and can beincorporated as a system in existing clinical chemistry equipment.Described specifically, a measurement sample is set on a measurementsystem. After the first enzyme is added to the measurement sample andacetaldehyde, phosphoric acid and ammonia are formed, at least one ofthe acetaldehyde, phosphoric acid and ammonia is quantified. As thismethod involves no reliance on human subjective view, it is suited forroutine processing by the measurement system and can lead to quickly andsimply measuring a large number of samples. Such processing can also beperformed by making a computer, which is incorporated in the measurementsystem, read a program that allows the measurement system to conduct thefirst step of adding the enzyme to each measurement sample and thesecond step of quantifying at least one of acetaldehyde, phosphoric acidand ammonia.

As described above, there is a significant correlation between theconcentration of ethanolamine phosphate in plasma and depression (seePatent Document 3). By further adding, to the above-described program, astep of determining, based on the measurement value of ethanolaminephosphate level in a measurement sample, whether or not the subject whoprovided the measurement sample is a depressive patient and another stepof outputting the measurement result so obtained, it is hence possibleto diagnose whether or not the subject who provided the measurementsample is a depressive patient.

The program maybe recorded on a recording medium readable by a computer,or on a recording medium in a computer attached to the measurementsystem. The recording medium may be, but is not limited specifically to,a hard disk, CD, DVD, USB memory, Floppy® disk, or the like.

According to the present invention, there is also provided anethanolamine phosphate-measuring reagent which contains an enzymecapable of catalyzing a reaction that forms acetaldehyde fromethanolamine phosphate. As the enzyme capable of catalyzing the reactionthat forms acetaldehyde from ethanolamine phosphate, it is possible touse an enzyme similar to that employed in the first step of themeasurement method of the ethanolamine phosphate level. Describedspecifically, proteins consisting of the amino acid sequences describedunder SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing can bementioned.

Preferably, the ethanolamine phosphate-measuring reagent may furthercontain a coenzyme for the enzyme to further improve the react ionefficiency of the enzyme. As a specific example of the coenzyme,pyridoxal phosphate can be mentioned. No particular limitation isimposed on the enzyme insofar as it can catalyze the reaction of theequation (1) that hydrolyzes ethanolamine phosphate to formacetaldehyde, phosphoric acid and ammonia in one step. As an example ofthe enzyme that has the enzyme activity, an enzyme having a GabT domaincan be mentioned. As an alternative, the enzyme may be a protein havingan amino acid sequence region having at least 90% of homology with theabove-described GabT domain.

Since there is a significant correlation between the concentration ofethanolamine phosphate in plasma and depression as described above, theethanolamine phosphate-measuring reagent can be used as a depressiondiagnostic agent.

According to the present invention, there is also provided anethanolamine phosphate-measuring kit comprising a container with anenzyme, which can catalyze a reaction that forms acetaldehyde fromethanolamine phosphate, separately contained therein. Because thesuffering from depression can be determined from the concentration ofethanolamine phosphate in plasma as described above, the use of theethanolamine phosphate-measuring kit according to the present inventionmakes it possible to conduct screening of depressive patients from thosesubjected to an examination or check-up under a physical examinationprogram or employees' in-house medical check-up program, to allow anon-specialist physician to conduct a diagnosis of a patient withsuspected depression, or to allow a specialist to conduct a diagnosisalong with an interview, to assess the effectiveness of treatment and todetermine a strategy of treatment.

Preferably, the ethanolamine phosphate-measuring kit may furthercomprise another container with a coenzyme for the enzyme, said coenzymebeing contained separately in said another container. As an example ofthe coenzyme, pyridoxal phosphate can be mentioned. No particularlimitation is imposed on the above-described enzyme insofar as it cancatalyze the reaction of equation (1) that forms acetaldehyde,phosphoric acid and ammonia in one step. As an example of the enzymehaving the enzyme activity, an enzyme having a GabT domain can bementioned. As an alternative, the enzyme maybe a protein having an aminoacid sequence region having at least 90% of homology with theabove-described GabT domain.

EXAMPLES

1. Preparation of Cell Lysate with Ethanolamine Phosphate-degradingEnzyme Contained Therein

As cell strains having ethanolamine phosphate lyase activity, threeErwinia strains (Erwinia carotovora, Pantoea ananatis LMG 20103 strain,and Erwinia milletiae) were selected, and were purchased fromIncorporated Administrative Agency, National Institute of AgrobiologicalSciences. The three strains were subjected to liquid culture in portionsof an oligotrophic synthetic medium with ethanolamine contained as asingle nitrogen source therein. Resulting colonies were spread on LBplates and cultured overnight at 30° C., and colonies were obtained onthe respective plates.

From each plate, two to three colonies were harvested, suspended in aliquid medium (2 to 5 mL), and cultured at 30° C. and 170 rpm. Aspronounced proliferation was observed only on Pantoea ananatis out ofthe above-described three strains, that cell strain was cultured untilOD₆₀₀=1.0, and subsequent to the culture, centrifugation was conductedto harvest its cells.

The thus-obtained cell strain was suspended in Buffer A [a solution of2-mercaptoethanol (final concentration: 1 mM) in tris-hydrochloric acidbuffer (final concentration: 50 mM), pH 7.5], cell disruption wasconducted by sonication, and subsequent to centrifugation, a supernatantwas collected to prepare a lysate (hereinafter called “the Pan lysate”).

2. Measurement of Ethanolamine Phosphate-degrading Enzyme Activity ofPan Lysate

Referential Example 1

To a reaction solution (1 mL) containing ethanolamine phosphate (finalconcentration: 2 mM) as a reaction substrate and pyridoxal phosphate(final concentration: 2 mM) as a coenzyme, the Pan lysate was added(final volume ratio: 5%), and the resulting mixture was incubated at 30°C. One hour later, the mixture (200 μL) was deproteinized by filtration(“MICROCON® (a centrifugal filter), Mw: 5k) to terminate the reaction.Subsequently, the concentration of ethanolamine phosphate in thefiltrate was measured using a quadropole mass spectrometer (“QuadropoleLC/MS 6140” manufactured by Agilent Technologies, Inc.). A similarmeasurement was conducted on the sample after 4 hours and 20 hours fromthe initiation of the experiment. Results are indicated by black dots(●) in FIG. 1.

Referential Example 2

Measurements were conducted as in Referential Example 1 except for theuse of a 10-fold dilute solution of the Pan lysate in Buffer A in placeof the Pan lysate. Results are indicated by black triangles (▴) in FIG.1.

Referential Example 3

Measurements were conducted as in Referential Example 1 except for theuse of a 100-fold dilute solution of the Pan lysate in Buffer A in placeof the Pan lysate. Results are indicated by black rhombi (♦) in FIG. 1.

Referential Example 4

Measurements were conducted as in Referential Example 1 except for theuse of Buffer A, as a negative control, in place of the Pan lysate.Results are indicated by black squares (▪) in FIG. 1.

From the results shown in FIG. 1, it is evident that the ethanolaminephosphate in each measurement sample was degraded and decreased inconcentration with time by the enzyme in the Pan lysate. It was,therefore, demonstrated that an ethanolamine phosphate-degrading enzymewas contained in the Pan lysate.

3. Isolation of Ethanolamine Phosphate Lyase Derived from Pantoeaananatis

With the same culture medium and under the same conditions as in Item 1described above, Pantoea ananatis was subjected to mass culture on theliquid medium. The thus-obtained cells were suspended in Buffer B [asolution of magnesium chloride (final concentration: 10 mM) intris-hydrochloric acid buffer (final concentration: 20 mM), pH 7.5],cell disruption was conducted by sonication, and then, centrifugationwas performed to obtain a supernatant fraction. The fraction was firstsubjected to elution fractionation under a gradient of sodium chloride(100-500 mM) by anion chromatography (“DE-52”, product of GE HealthcareJapan Corporation), and then, SDS-PAGE was conducted on respectivefractions. The respective fractions were added to portions of atris-hydrochloric acid buffer (final concentration: 20 mM; pH 7.5),which contained ethanolamine phosphate (final concentration: 1 mM) andpyridoxal phosphate (final concentration: 1 mM), such that the finalvolume ratios of the fractions reached 30%. After the resulting mixtureswere separately subjected to the reaction at 30° C., acetaldehydedehydrogenase (ALDH) and oxidized nicotinamide dehydrogenase (NAD⁺) wereadded to the respective samples after the reaction according to themanual of a commercial acetaldehyde quantification kit (“F-kitAcetaldehyde”, product of Roche Diagnostics K.K.). Subsequent to thereaction of equation (2), the absorbances at 340 nm were measured. Fromeach absorbance, the acetaldehyde in the corresponding sample after thereaction of equation (1) was quantified to determine the ethanolaminephosphate-degrading enzyme activity in the sample before the reaction ofequation (1). The results of SDS-PAGE and the measurement results ofethanolamine phosphate-degrading enzyme activity are shown in an upperpart and lower part of FIG. 2, respectively, such that each fraction inthe upper graph and its corresponding fraction in the lower graph arealigned in the same column. The numbers in the lowest row are collectedfraction numbers. “ft” means a flow-through, “w” denotes a wash solution(Buffer C-1) containing sodium chloride at 100 mM in Buffer B, and “wo”signifies awash solution (Buffer C-2) containing sodium chloride at 500mM in Buffer. As shown in FIG. 2, strong ethanolaminephosphate-degrading enzyme activity was confirmed on the collectedfractions numbered from 12 to 18. Further, it was considered from theresults of SDS-PAGE that the bands around 75 kDa, 45 kDa and 40 kDa werelikely those of ethanolamine phosphate-degrading enzymes.

The fractions numbered from 12 to 18 were then combined together, andthe resulting solution was subjected to elution fractionation underammonium sulfate (1-0 M) by hydrophobic chromatography (“Ether-650”,product of Tosoh Corporation). As eluents, Buffer B and a solution(Buffer D-1) containing ammonium sulfate at 1 M in Buffer B were used.Similar to the above-described anion chromatography, SDS-PAGE andabsorbance measurements using the acetaldehyde quantification kit wereconducted on the respective fractions. Results are shown in FIG. 3. Asshown in FIG. 3, high ethanolamine phosphate-degrading enzyme activitywas confirmed on the collected fractions numbered from 7 to 10. Further,it was considered from the results of SDS-PAGE that the bands around 75kDa, 45 kDa and 25 kDa were likely those of ethanolaminephosphate-degrading enzymes.

The fractions numbered from 7 to 10 in the above-described hydrophobicchromatography were collected, and were subjected to fractionationthrough a hydroxyapatite column (“HT-Gel”, product of Bio-RadLaboratories, Inc.). As eluents, a 10 mM potassium phosphate solution(pH 9.0) (Buffer E-1) and a 300 mM potassium phosphate solution (pH 9.0)(Buffer E-2) were used. Similar to the above-described anionchromatography, SDS-PAGE and absorbance measurements using theacetaldehyde quantification kit were conducted on the respectivefractions. Results are shown in FIG. 4. As apparent from FIG. 4, highethanolamine phosphate-degrading enzyme activity was confirmed on thecollected fractions of numbers w and 1. Further, it was considered fromthe results of SDS-PAGE that the bands around 45 kDa and 25 kDa werelikely those of ethanolamine phosphate-degrading enzymes.

From the results of the fractionation by the three kinds ofchromatography, the three bands of 75 kDa, 45 kDa and 25 kDa wereselected as candidates for ethanolamine phosphate-degrading enzymes.

In each of the experiments described above, acetaldehyde was formed bythe addition of the cell lysate of Pantoea ananatis to the solution thatcontained ethanolamine phosphate. It has, therefore, been confirmed thatthe ethanolamine phosphate-degrading enzymes described above are enzymescapable of catalyzing the reaction of equation (1).

4. Identification of Ethanolamine Phosphate Lyase Derived from Pantoeaananatis

The three bands selected in Item 3 described above were collected fromthe SDS-PAGE gel, and the determination of their amino acid sequenceswas contracted to a custom service provider (Hokkaido System ScienceCo., Ltd.). As a result, those bands were all recognized to have highhomology with the below-described proteins, respectively, and from theirmolecular weights and N-terminal amino acid sequences, were alsoconfirmed to correspond to the following proteins, respectively.

-   -   75 kDa: phenylalanyl-tRNA synthesis enzyme, β-subunit (Pantoea        vagans C9-1)    -   45 kDa: GabT (Pantoea ananatis LMG20103) shown as SEQ ID NO:1    -   25 kDa: WrbA (Pantoea ananatis LMG20103)

Among the above-described proteins, the phenylalanyl-tRNA synthesisenzyme and WrbA have been reported to catalyze reactions different fromdeamination and dephosphorylation. They were, accordingly, excluded fromthe candidates for ethanolamine phosphate-degrading enzymes.

The remaining gene for GabT has been confirmed to exist in the genomesof a relatively small number of microorganism species, plants, andanimals such as human. In animals, its expression in the brain has beenreported. As a function of GabT, GabT is known to transfer an aminogroup from γ-aminobutyric acid (GABA) as a substrate to an organic acidin a pyridoxal phosphate-dependent manner.

Further, GabT has also been reported to have transamination(deamination) activity for a relatively broad range of substrates otherthan GABA, and with GabT derived from E. coli, transamination activityfor 3-aminopropyl(methyl)phosphinic acid similar in structure toethanolamine phosphate has been reported. The ethanolamine phosphatedegradation reaction of the equation (1) is accompanied bydephosphorylation, and therefore, is different from known reaction modesof GabT. In view of the above-described similarity, however, GabT hasbeen determined to have a high possibility of being an enzyme thatcatalyzes the reaction of equation (1).

5. Cloning of Pantoea ananatis gabT Gene and E. coli gabT Gene

A band corresponding to the gabT gene was obtained from the genome ofPantoea ananatis by a known PCR-dependent cloning method, and wasligated to pUC18 for amplification. A DNA sequence analysis wascontracted to a custom service provider (Hokkaido System Science Co.,Ltd.), and the target gabT gene was confirmed to be obtained. The aminoacid sequence of the resulting Pantoea ananatis-derived GabT is shown asSEQ ID NO:1 in the accompanying sequence listing.

Similarly, from E. coli (wt) in the genome of which the existence of thegabT gene has been confirmed, the genome was extracted, a bandcorresponding to the gabT gene was obtained by a known cloning method,and the band was ligated to pUC18 for amplification. As a result of aDNA sequence analysis, the target gabT gene was confirmed to beobtained. The amino acid sequence of the resulting E. coli-derived GabTis shown as SEQ ID NO:2 in the accompanying sequence listing, and thebase sequence of the E. coli-derived gabT gene is shown as SEQ ID NO:3.

6. Construction of GabT Expression System and Confirmation of EnzymeActivity

Referential Example 5

The Pantoea ananatis-derived gabT gene, an expression product of whichwill be abbreviated as P-GabT″, was inserted in the expression vectorpET23a to transform the E. coli BL21(DE3) strain. The transformed E.coli was cultured in LB medium, and IPTG was added in a presteady stateto induce the expression of P-GabT. Five hours after the induction,centrifugation was conducted to harvest cells. The thus-obtained cellswere suspended in a lysis buffer with pyridoxal phosphate containedtherein (10 mM pyridoxal phosphate, 100 mM potassium chloride, 1 mMethylenediaminetetraacetic acid, 100 mM potassium chloride, 1 mMdithiothreitol, 50 mM tris-hydrochloric acid, pH 7.5), and then, celldisruption was conducted by sonication.

The resulting cell lysate was centrifuged to collect a supernatantfraction. The cell lysate before the centrifugation, which will beabbreviated as “total”, and the supernatant fraction, which will beabbreviated as “sup”, were separately added to portions of atris-hydrochloric acid buffer (final concentration: 20 mM: pH 7.5)containing ethanolamine phosphate (final concentration: 1 mM) andpyridoxal phosphate (final concentration: 1 mM) such that their finalvolume ratios reached 30%. After the resulting mixtures were separatelysubjected to the reaction at 30° C., acetaldehyde dehydrogenase (ALDH)and oxidized nicotinamide dehydrogenase (NAD⁺) were added to therespective samples after the reaction according to the manual of thecommercial acetaldehyde quantification kit (“F-kit Acetaldehyde”,product of Roche Diagnostics K.K.). Subsequent to the reaction ofequation (2), the absorbances at 340 nm were measured. From eachabsorbance, the acetaldehyde in the corresponding sample after the reaction of equation (1) was quantified, and the ethanolaminephosphate-degrading enzyme activity in the sample before the reaction ofequation (1) was determined on the basis of acetaldehyde level. Resultsare indicated by “P-” in FIG. 5. In the graph, the ordinate indicatesthe absorbance at 340 nm, and each error bar indicates a standarddeviation obtained from thrice repeated measurements.

Referential Example 6

By similar methods and procedures as in Referential Example 5 except forthe use of E. coli-derived gabT gene (an expression product of whichwill be abbreviated as “E-GabT”) in place of the Pantoeaananatis-derived gabT gene, an experiment was conducted to obtain a celllysate and supernatant fraction. Their ethanolamine phosphate-degradingenzyme activities were measured. Results are indicated by “E-” in FIG.5.

Referential Example 7

By similar methods and procedures as in Referential Example 5 exceptthat the transformation was conducted using pET23a alone as a negativecontrol, an experiment was conducted. The ethanolaminephosphate-degrading enzyme activities of a cell lysate and supernatantfraction were measured. Results are indicated by “NC” in FIG. 5.

From the results of Referential Examples 5 to 7, in the case of the celllysates on a left side in FIG. 5, it was possible to confirm highethanolamine phosphate-degrading enzyme activity on both P-GabT andE-GabT compared with the negative control. In the case of thesupernatant fractions on a right side in FIG. 5, on the other hand, bothP-GabT and E-GabT had similar enzyme activity as the negative control.Further, also in SDS-PAGE, P-GabT and E-GabT were both confirmed as darkbands at 45 kDa in the case of the cell lysates, but were of similarlevels as the control in the case of the supernatant fractions (notshown). From the above results, P-GabT and E-GabT are both considered toexist as inclusion bodies in the precipitate fractions.

7. Determination of Sequences of GabT Domains

Referential Example 8

The amino acid sequences of GabTs (aminobutyric acid aminotransferase inthe case of mammals) derived from Sulfolobus (Sulfolobus tokodaii str.7; SEQ ID NO: 4) as an archaebacteria, human (Homo sapiens; SEQ ID NO:5) and mouse (Mus musculus; SEQ ID NO: 6) as the mammals, andArabidopsis (Arabidopsis thaliana; SEQ ID NO: 7) as a plant, in additionto Pantoea ananatis and E. coli used in Referential Examples 5 to 7,were aligned using a commercial program (“CLC Sequence Viewer”, preparedby CLC Bio Japan, Inc.). Results are shown in FIG. 6. In FIG. 6, theregion boxed by a thick line and containing the 301^(st) to 317^(th)amino acids was extracted as a region of high conservation. It is to benoted that the amino acids shown in rows labeled “consensus” areresidues conserved 60% or more.

Referential Example 9

In addition to the GabT sequences of the above-described six organismspecies, Salmonella (Salmonella enterica subsp. enterica serovar Typhistrain CT18), Clostridium (Clostridium acetobutylicum ATCC 824),Pseudomonas (Pseudomonas putida KT2440), Rhodococcus (Rhodococcus equi103S), Acinetobacter (Acinetobacter baumanii ACICU), Mycobacter(Mycobacterium avium subsp. paratuberculosis K-10), cattle (Bos taurus),larvacea (Oikopleura dioica), maize (Zea mays) and red pepper (Capsicumannuum) were also verified for the conservation of the domain regionsspecified in Referential Example 8. Concerning the amino acid sequenceranging from the 301^(st) to the 317^(th), at least 90% of homology wasconserved in the above-described 16 organism species. This region was,therefore, defined as a GabT domain.

The GabT domain is an amino acid sequence region consisting of thefollowing amino acid sequence:

GXXXBADEBQXGFZRXGwherein X means an amino acid, B denotes a branched amino acid selectedfrom the group consisting of I, L and V, and Z signifies G or A. Thesequences of the GabT domains of the respective organism species areshown in Table 1. In the sequences of Table 1, those different from thecorresponding amino acids in the GabT domain defined as described aboveare indicated by underlining them.

TABLE 1 Homology Organism name/organism species Sequence (%) SEQ ID NO:Pantoea / Pantoea ananatis LMG 20103 GIMLICDEIQSGFGRTG 91 8E. coli / Escherichia coli str. K-12 substr. GIMLIADEVQSGAGRTG 91 9MG1655 Sulfolobus / Sulfolobus tokodaii str. 7 GILLVDDEVQMGLGRTG 91 10Salmonella / Salmonella enterica subsp. GIMLIADEVQSGAGRTG 91 11enterica serovar Typhi str. CT18Clostridium / Clostridium acetobutylicum ATCC DIVFIIDEVQAGFGRTG 91 12824 Pseudomonas / Pseudomonas putida KT2440 GILLIADEVQTGAGRTG 91 13Rhodococcus / Rhodococcus equi 103S GIVFVADEVQTGFARTG 100 14Acinetobacter / Acinetobacterbaumannii ACICU GILLVADEVQSGFARTG 100 15Mycobacter / Mycobacterium avium subsp. DVLFIADEVQTGFARSG 91 16paratuberculosis K-10 Human / Homo sapiens GGVFIADEVQVGFGRVG 100 17Bovine / Bos taurus GGVFIADEVQVGFGRVG 100 18 Mouse / Mus musculusGGLFVADEIQVGFGRIG 100 19 Ascidian / Oikopleura dioica GVLTIADEVQVGFGRVG100 20 Thale cress / Arabidopsis thaliana GGVCIADEVQTGFGRTG 100 21Corn / Zea mays GGLCIADEVQAGFARVG 100 22 Cayenne / Capsicum annuumGGVCIADEVQTGFGRTG 100 23

The invention claimed is:
 1. A composition for screening a human patienthaving depression, comprising: a plasma sample of a human subject; andan ethanolamine phosphate-measuring reagent comprising an enzyme thatcatalyzes a reaction that forms acetaldehyde from ethanolaminephosphate, wherein the enzyme is a protein having an amino acid sequenceregion having at least 90% of homology with a GabT domain consisting ofa following amino acid sequence: GXXXBADEBQXGFZRXG

wherein X means an amino acid, B denotes a branched amino acid selectedfrom the group consisting of I, L, and V, and Z signifies G or A, andthe enzyme is derived from at least one source material selected fromthe group consisting of Pantoea, E. coli, Sulfolobus, Arabidopsis,Salmonella, Clostridium, Pseudomonas, Rhodococcus, Acinetobacter,Mycobacter, cattle, mice, larvacea, maize, and red peppers.
 2. Thecomposition according to claim 1, wherein the enzyme is: (1) a proteinconsisting of an amino acid sequence represented by SEQ ID NO:1 or 2,(2) a protein consisting of an amino acid sequence that in the aminoacid sequence represented by SEQ ID NO:1 or 2one or a few amino acidshave been deleted, substituted, or added, or (3) a protein consisting ofan amino acid sequence having at least 90% of homology with the aminoacid sequence represented by SEQ ID NO:1 or
 2. 3. The compositionaccording to claim 1, further comprising a coenzyme for the enzyme. 4.The composition according to claim 3, wherein the coenzyme is pyridoxalphosphate.
 5. The composition according to claim 1, wherein the enzymeis derived from Pantoea ananatis.
 6. A composition for determiningwhether a human patient has depression, comprising: a plasma sample ofthe human patient; and an ethanolamine phosphate-measuring reagentcomprising an enzyme that catalyzes a reaction that forms acetaldehydefrom ethanolamine phosphate, wherein the enzyme is a protein having anamino acid sequence region having at least 90% of homology with a GabTdomain consisting of a following amino acid sequence: GXXXBADEBQXGFZRXG

wherein X means an amino acid, B denotes a branched amino acid selectedfrom the group consisting of I, L, and V, and Z signifies G or A, andthe enzyme is derived from at least one source material selected fromthe group consisting of Pantoea, E. coli, Sulfolobus, Arabidopsis,Salmonella, Clostridium, Pseudomonas, Rhodococcus, Acinetobacter,Mycobacter, cattle, mice, larvacea, maize, and red peppers.
 7. Thecomposition according to claim 6, wherein the enzyme is derived fromPantoea ananatis.